title: Agitation during lipoplex formation improves the gene knockdown effect of siRNA.
abstract: The successful delivery of therapeutic siRNA to the designated target cells and their availability at the intracellular site of action are crucial requirements for successful RNAi therapy. In the present study, we focused on the siRNA-lipoplex preparation procedure and its effect on the gene-knockdown efficiency of siRNA in vitro. Agitation (vortex-mixing) during siRNA-lipoplex (vor-LTsiR) preparation and its effect on the gene-knockdown efficiency of stably expressed cell GFP was investigated, and their efficiency was compared with that of spontaneously formed lipoplex (spo-LTsiR). A dramatic difference in size between lipoplexes was observed at the N/P ratio of 7.62 (siRNA dose of 30 nM), even though both lipoplexes were positively charged. With the siRNA dose of 30 nM, vor-LTsiR accomplished a 50% gene-knockdown, while spo-LTsiR managed a similar knockdown effect at the 120 nM level, suggesting that the preparation procedure remarkably affects the gene-knockdown efficacy of siRNA. The uptake of vor-LTsiR was mainly via clathrin-mediated endocytosis, whereas that of spo-LTsiR was via membrane fusion. In addition, by inhibiting clathrin-mediated endocytosis, the gene-knockdown efficiency was significantly lowered. The size of the lipoplex, promoted by the preparation procedure, is likely to define the entry pathway, resulting in an increased amount of siRNA internalized in cells and an enhanced gene-knockdown efficacy. The results of the present study definitively show that a proper siRNA-lipoplex preparation procedure makes a significant contribution to the efficiency of cellular uptake, and thereby, to the gene-knockdown efficiency of siRNA.
authors.affiliation: Department of Pharmacokinetics and Biopharmaceutics, Institute of Health Biosciences, The University of Tokushima, 1-78-1 Sho-machi, Tokushima, Japan.
authors.forename: Jose Mario
authors.initials: JM
authors.lastname: Barichello
authors.forename: Shinji
authors.initials: S
authors.lastname: Kizuki
authors.forename: Tatsuaki
authors.initials: T
authors.lastname: Tagami
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Asai
authors.forename: Tatsuhiro
authors.initials: T
authors.lastname: Ishida
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kikuchi
authors.forename: Naoto
authors.initials: N
authors.lastname: Oku
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kiwada
mesh_terms: D002412:Cations; D045744:Cell Line, Tumor; D002966:Clathrin / Q000378:metabolism; D004705:Endocytosis; D055785:Gene Knockdown Techniques / Q000379:methods*; D020868:Gene Silencing; D006801:Humans; D008055:Lipids / Q000737:chemistry*; D008081:Liposomes; D034741:RNA, Small Interfering / Q000008:administration & dosage*
chemical_list: D002412:Cations; D002966:Clathrin; D008055:Lipids; D008081:Liposomes; D034741:RNA, Small Interfering